**2. Results**

### *2.1. Batch Fermentations and Production of Value-Added Compounds by Yarrowia lipolytica Growing on OMW*/*Glycerol Blends Supplemented with NaCl*

Experiments were carried out investigating the assimilation of glycerol and its conversion by the yeas<sup>t</sup> *Y. lipotytica* ACA-YC 5031 to secondary metabolites with and without the addition of OMWs (phenolic compounds into the media = 2.0 ± 0.20 g/L) into the medium. The e ffect on the microbial reactions and physiology of di fferent concentrations of NaCl (1.0%, 3.0%, and 5.0% *w*/*v*) added into the culture medium was also examined. Under all circumstances, there was production of secondary metabolites, such as SCO, citric acid, intracellular polysaccharides (IPS), and polyols (mainly mannitol and erythritol, while small concentrations of arabitol were also detected) during growth on OMW/glycerol blends, where variable quantities of NaCl were added. The secretion of extra-cellular metabolites was enhanced after virtual nitrogen depletion from the growth medium occurring *c*. 24–30 h after inoculation. Specifically, the initial nitrogen concentration of nitrogen as indicated by the concentration of extra-cellular free amino nitrogen (FAN) was = 85 ± 10 mg/L, whereas the final nitrogen concentration when this compound became the limiting substrate was 25 ± 5 mg/<sup>L</sup> of FAN into the medium (data not presented). The results of the maximum values of the components obtained during the fermentations carried out are presented in Table 1.


**Table 1.** values of glycerol consumption, production of biomass, cellular lipids, citric acid, mannitol, erythritol, intra-cellular polysaccharides (IPS) and yield coefficients in culture medium with just glycerol (blank) and glycerol blended with OMWs yielding at initial concentration of 2.0 g/<sup>L</sup> of phenols using different concentrations of NaCl *w*/*v* (0.0%, 1.0%, 3.0%, 5.0%).

In bold we highlight the maximum value of each fermentation compound (i.e. biomass, citric acid, etc) for each trial performed, whereas with bold and underline we highlight the maximum quantity of the given compound between alltrials.

The results showed a small reduction of biomass (dry cell weight – DCW; g/L) production and a net reduction of polyols biosynthesis as well as reduced glycerol consumption after the addition of OMWs (*viz.* phenolic compounds) and NaCl into the culture medium, while concomitantly, citric acid (*Cit*) production significantly increased. Potentially, the addition of toxic compounds (i.e. OMWs and NaCl) slightly negatively affected biomass production and substrate assimilation, in accordance with several reports in the literature [3,8,11]. Moreover, it was observed that the addition of NaCl combined with the addition of phenolic compounds into the medium favored the yeas<sup>t</sup> to increase the production of lipids in its biomass (*YL*/*X* in % *w*/*w*). Apparently, the combined effect of the high osmotic pressure and the addition of a "lipogenic" medium, such as the OMWs [3,8], increased the quantity of lipid accumulated inside the yeas<sup>t</sup> cells. In a restricted number of cases, the addition of several types of natural substances (i.e. phenolic compounds, essential oils, etc.) has been revealed to be capable of increasing the content of cellular fatty acids (FAs) inside *Y. lipolytica* yeas<sup>t</sup> [3,8,43], similarly to the current investigation. More specifically, the yeas<sup>t</sup> consumed all glycerol without the presence of OMWs and NaCl in the culture medium after 260 h of fermentation, obtaining the highest DCW value between all the experiments (10.8 g/L) with biomass yield (*YX*/*Glol*) = 0.14 g/g and endopolysaccharides (IPS) = 0.88 g/<sup>L</sup> (corresponding to polysaccharides per DCW value (*YIPS*/*X)* = 8.1% *w*/*w*). However, during this fermentation, maximum production of intracellular lipids, 2.39 g/L, with maximum lipid in DCW value (*YL*/*X*) = 23.9% *w*/*w* was observed earlier, at 188 h, at the same time producing the maximum value of citric acid 10.5 g/<sup>L</sup> with *YCit*/*Glol* = 0.15 g/g. At the same conditions (no OMWs and no NaCl added), the highest values of mannitol (13.4 g/<sup>L</sup> at 188 h) and erythritol (8.8 g/<sup>L</sup> at 164 h) were obtained, and these values corresponded to the highest ones obtained in all trials carried out. The addition of OMWs yielding in initial phenolic compounds concentration of *c*. 2.0 g/<sup>L</sup> in the culture medium slightly reduced the produced biomass (*Xmax* = 8.7 g/L) and the assimilation rate of glycerol. On the other hand, the amount of the maximum produced intracellular lipids and IPS were slightly increased, while citric acid production was significantly higher compared to the trial with no OMWs added (see Table 1). More specifically, maximum production of polyols (8 g/<sup>L</sup> mannitol with *YMan*/*Glol* = 0.16 g/g and 4.9 g/<sup>L</sup> erythritol with *YEry*/*Glol* = 0.10 g/g) was observed at 140 h of fermentation. Maximum production of IPS, 1.52 g/<sup>L</sup> corresponding to 17.5% of the produced biomass, and 32.7 g/<sup>L</sup> of citric acid with *YCit*/*Glol* = 0.52 g/g were obtained at 188 h. At the same time, *YL*/*X* = 28.3% *w*/*w* was obtained. Finally, up to 93% glycerol consumption was observed. Similar results were observed when 1.0% NaCl was added, while the highest values of intracellular lipids and IPS were obtained compared to all fermentations. More specifically, 2.52 g/<sup>L</sup> intracellular lipids were produced at 242 h with an increased percentage of produced lipids per produced biomass ( *YL*/*X* = 29.9% *w*/*w*). The maximum produced IPS was 2.59 g/<sup>L</sup> at 224 h. At the same time, the maximum production of citric acid, 45.2 g/L, and mannitol, 6.0 g/L, were obtained, with conversion yields of 0.68 and 0.09 g/g, respectively. Maximum produced erythritol was just 2.5 g/<sup>L</sup> at 144 h with *YEry*/*Glol* = 0.05 g/g.

The increased concentration of NaCl to 3.0% in the culture medium resulted in reduced glycerol consumption (up to 76%) and decreased maximum DCW and polyols production. Although the absolute values of produced biomass and intracellular lipids were decreased, the maximum lipid in DCW value *YL*/*X* (% *w*/*w*) was increased to 31.5% at 280 h. Maximum value of DCW, 5.6 g/L, was observed at 140 h having, however, the highest produced biomass per consumed substrate between the fermentations ( *YX*/*Glol* = 0.16 g/g). At the same time, maximum production of erythritol was 4.9 g/<sup>L</sup> with a simultaneous yield 0.14 g/g. Maximum concentration of IPS was decreased to 0.72 g/<sup>L</sup> at 280 h, corresponding to endopolysaccharides in DCW = 19.8% *<sup>w</sup>*/*<sup>w</sup>*. Maximum produced citric acid and mannitol were observed at 280 h, with values 42.0 and 3.6 g/L, respectively, suggesting the positive combined e ffect of NaCl and OMWs addition upon the shift of the cellular metabolism toward citric acid production.

The addition of 5.0% *w*/*v* of NaCl favored the growth of the yeast, the assimilation of glycerol, and the production of secondary metabolites more than with the addition of 3.0% *w*/*v* NaCl, with the exception of polyols production. At these conditions, the percentage of lipids in produced biomass was further increased, reaching the highest value of 35.1% *w*/*w* with a 2.25 g/<sup>L</sup> maximum of produced intracellular lipid after 308 h of fermentation. At that time, the maximum glycerol consumption was 90% *<sup>w</sup>*/*<sup>w</sup>*, and the maximum erythritol production was only 3.2 g/L. Maximum DCW concentration was 6.8 g/L, with a yield *YX*/*Glol* = 0.10 g/g at 233 h, whereas, at the same time, the highest concentration of citric acid (54.0 g/<sup>L</sup> with simultaneous yield *YCit*/*Glol* = 0.82 g/g) was achieved. Those conditions favored the yeas<sup>t</sup> to produce citric acid in the best conversion yield of citric acid produced per glycerol consumed of all conversions carried out (global conversion yield of citrate produced per unit of glycerol consumed = 0.81 g/g; see Figure 1).

**Figure 1.** Citric acid production vs glycerol consumption by *Yarrowia lipolytica* ACA-YC 5031 in 70.0 g/<sup>L</sup> glycerol (blank) and glycerol blended with 2.0 g/<sup>L</sup> phenolic compounds and different concentrations of NaCl (0.0%, 1.0%, 3.0%, and 5.0% *w*/*v*).

Figure 1 illustrates the correlation between glycerol consumption and citric acid production, and clearly, the more NaCl is added, the more *YCit*/*Glol* increases. The absence of phenolic compounds and NaCl from the culture medium (blank experiment) resulted in much less citric acid production per consumed glycerol (≈0.11 g/g). Citric acid volumetric productivity was 0.05 g/(L·h), with a maximum value 10.5 g/L. While all substrate (glycerol) was consumed with a consumption rate 0.40 g/(L·h), a significant number of polyols was produced (20.0 g/L) at that time, also producing a high amount of biomass (Figure 2). The addition of the phenolic compounds (2.0 ± 0.20 g/L) into the medium resulted in 94% glycerol consumption, with a consumption rate 0.32 g/(L·h) and a bit less biomass (*Xmax* = 8.7 g/L) compared to the previous fermentation. A clear decrease in polyols production was also observed, while the productivity of citric acid was faster (0.18 g/L·h), and a significantly increased value, up to 32.7 g/L, was observed (Figure 2b). The kinetics of glycerol (*Glol*) consumption, biomass (*X*) production, polyols biosynthesis (Σ*Polyols*), and citric acid (*Cit*) accumulation into the growth medium for all trials performed is shown in Figure 2a–e. Likewise, the illustration of parameters concerning the trials performed is presented in Table 2.

**Figure 2.** Kinetics of glycerol consumption (g/L), yeas<sup>t</sup> *Yarrowia lipolytica* ACA-YC 5031 growth (DCW g/L), citric acid, and polyols production (g/L) in a glycerol-based culture medium containing (**a**) phenols = 0.0 g/<sup>L</sup> and NaCl = 0.0%; (**b**) phenols = 2.0 g/<sup>L</sup> and NaCl = 0.0%; (**c**) phenols = 2.0 g/<sup>L</sup> and NaCl = 1.0%; (**d**) phenols = 2.0 g/<sup>L</sup> and NaCl = 3.0% and (**e**) phenols = 2.0 g/<sup>L</sup> and NaCl = 5.0% concentration.


**Table 2.** Glycerol consumption (%), glycerol consumption rates, citric acid production rates, and specific citric acid production rates during fermentations of glycerol (70.0 g/L) with and without phenolic compounds (2 g/L) and with increased concentrations of NaCl (0.0%, 1.0%, 3.0% and 5.0% *w*/*v*).

### *2.2. Intra-Cellular Lipid Concentration and Fatty Acid Composition*

The intra-cellular lipid concentration was in most of the cases steadily increased during all fermentations (Figure 3). However, the combined e ffect of the addition of OMWs and NaCl into the medium increased lipid in DCW values of *Y. lipolytica* ACA-YC 5031 (without OMWs and NaCl, *YL*/*Xmax* value was 23.9% *<sup>w</sup>*/*<sup>w</sup>*, while with the addition of OMWs and NaCl at 5%, the respective value was 35.1% *w*/*w*—see Table 1). Specifically, the *YL*/*Xmax* value increased after the addition of OMW in the culture medium from 23.9% (blank experiment) to 28.3% (OMWs added; no NaCl added). *YL*/*Xmax* values continued to increase to 29.9%, 31.5%, and 35.1% with increment of NaCl concentration in the culture medium (1.0%, 3.0%, and 5.0% *w*/*v* NaCl, respectively) (see Table 1).

**Figure 3.** Single cell oil (SCO) production by *Yarrowia lipolytica* ACA-YC 5031 in culture medium containing blank glycerol and blended also with phenols and 0.0%, 1.0%, 3.0%, and 5.0% *w*/*v* NaCl.

The intracellular fatty acid (FA) composition was analyzed using gas chromatography (GC) and is presented in Table 3. The analyzed fatty acids were palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid. The yeas<sup>t</sup> produced a significant amount of C18:1, up to 66.0%, mainly without the presence of phenols and NaCl in the medium. The addition of phenolic compounds, as well as the increased concentration of NaCl, resulted in a slightly decreased concentration of oleic acid, in contrast to the production of palmitic acid, which it slightly increased from 10.0% to 16.0% *<sup>w</sup>*/*<sup>w</sup>*. Maximum concentration of palmitoleic was observed when phenols and 3.0% *w*/*v* NaCl were in the medium (12.0% and 15.0%), while a lower concentration of linoleic acid was observed in their absence (6.0%). The addition of phenols, as well as of 1.0% NaCl, resulted in the same concentration of linoleic acid, 11.0%, which was decreased in time. The same phenomenon was observed for 3.0% and 5.0% *w*/*v* NaCl, reaching 9.0% and the highest concentration of 21.0%, respectively. Finally, FA C18:0 was found in low concentrations in all trials performed.


**Table 3.** Fatty acid composition (%, *w*/*w*) of *Yarrowia lipolytica* ACA-YC 5031 when grown in a glycerol-based media (70.0 g/L) blended with OMWs (2.0 g/<sup>L</sup> of phenolic compounds), in which different concentrations of NaCl (0.0%, 1.0%, 3.0%, and 5.0% *w*/*v*) were added.

### *2.3. Color and Phenolic Compounds Removal*

*Y. lipolytica* ACA-YC 50231 efficiently removed color and phenolic compounds from the growth medium during most of the trials performed. There was a remarkable (≈40.0%) color reduction after 50 h of incubation in the presence of OMWs and 1.0% *w*/*v* of NaCl, followed by a 30.0% decolorization in 3.0% NaCl and 17.5% in 0.0% NaCl in the medium. The decolorization became slower following that incubation time. During the other time, only 13.0% color was removed between 250 and 300 h of incubation in the presence of a higher concentration (=5.0% *w*/*v*) of NaCl in the medium (Figure 4a). On the other hand, significant removal of phenolic compounds (maximum removal ranging between 52.0 – 62.0% *w*/*w* irrespective of the quantity of NaCl initially added into the medium) occurred in all trials performed (Figure 4b).

**Figure 4.** (**a**) Decolorization (%) and (**b**) Removal of phenolic compounds (%) from the culture medium when *Yarrowia lipolytica* ACA-5031 was grown in 0.0%, 1.0%, 3.0%, and 5.0% NaCl containing 2.0 g/<sup>L</sup> phenolic compounds.
